Antibody Identification

1. Antibody Identification Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435 School of Health Related Professions University of Mississippi Medical Center

2. The Basics….. As you recall, Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serum If antibodies are detected, they must be identified…

3. Why do we need to identify? Antibody identification is needed for transfusion purposes and is an important component of compatibility testing It will identify any unexpected antibodies in the patient’s serum If a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur

4. Key Concepts In blood banking, we test “knowns” with “unknowns” When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known)

5. Reagent RBCs Screening Cells and Panel Cells are the same with minor differences: Screening cells Antibody detection Sets of 2 or 3 vials Panel cells Antibody identification At least 10 vials per set

6. Antibody Panel vs. Screen An antibody panel is just an extended version of an antibody screen The screen only uses 2-3 cells:

7. Antibody Panel An antibody panel usually includes at least 10 panel cells:

8. Panel Group O red blood cells

9. Panel Each of the panel cells has been antigen typed (shown on antigram) + refers to the presence of the antigen 0 refers to the absence of the antigen

10. Panel An autocontrol should also be run with ALL panels

11. Panel The same phases used in an antibody screen are used in a panel

12. Antibody ID Testing A tube is labeled for each of the panel cells plus one tube for AC:

13. Antibody Panel steps After reagent cells and patient plasma or serum are added, several phases of testing are conducted on each tube: Immediate Spin (IS) LISS/37°C (10-15 min.) Wash X3 AHG Coombs’ control cells if indicated

15. Step 1: IS Phase Perform immediate spin (IS) and grade agglutination; inspect for hemolysis Record the results in the appropriate space as shown:

16. Step 2: LISS/37°C Phase 2 drops of LISS are added, mixed and incubated for 10-15 minutes Centrifuge and check for agglutination Record results

17. LISS/37°C Phase (example)

18. What do you do next?

19. Step 3: AHG Indirect Antiglobulin Test (IAT) – this tests whether or not antibodies in patient’s serum will react with RBCs in vitro (i.e., in the tube) To do this we use the Anti-Human Globulin reagent (AHG)..the green stuff! Polyspecific Anti-IgG Anti-complement

20. AHG Phase Wash cells 3 times with saline (manual or automated) Add 2 drops of AHG and gently mix Centrifuge Read Record reactions

21. AHG Phase

22. And don’t forget….

24. You have agglutination…now what?

25. Interpreting Antibody Panels There are a few basic steps to follow when interpreting panels “Ruling out” means crossing out antigens that did not react (neg result) Circle the antigens that are not crossed out Consider antibody’s usual reactivity Look for a matching pattern

26. Always remember:

27. Here’s an example… …let’s get to work

28. RULING OUT

29. CIRCLE ANTIGENS NOT ELIMINATED

30. CONSIDER USUAL REACTIVITY

31. LOOK FOR MATCHING PATTERN

32. Interpretation

33. Additional Considerations Again, it’s important to look at: Autocontrol Negative - alloantibody Positive – autoantibody or alloantibody to recently transfused cells (RBCs have a life span of 120 days) Phases IS – cold (IgM) 37° - cold (some have higher thermal range) or warm reacting AHG – warm (IgG) or complement…significant!! If there is a reaction in ALL cells…may be autoantibody Reaction strength 1 consistent strength – one antibody Different strengths – multiple antibodies or dosage DTR – delayed transfusion reaction (donor cells are sensitized with patient’s antibody)DTR – delayed transfusion reaction (donor cells are sensitized with patient’s antibody)

34. Considerations: Dosage Strength of reaction may be due to “dosage” If panel cells are homozygous, a strong reaction may be seen If panel cells are heterozygous, reaction may be weak or even non-reactive Panel cells that are heterozygous should not be crossed out because antibody may cause a reaction too weak to be seen

35. Considerations Matching the pattern Single antibodies usually show a pattern that matches one of the antigens (see previous panel example) Multiple antibodies are more difficult to match because they often show variable reaction strengths

36. Confirming the antibody To confirm the antibody identified from a panel, it is important to Use the rule of three Phenotype the patient’s RBCs

37. Rule of three The rule of three must be met to confirm the presence of the antibody A p-value = 0.05 must be observed This gives a 95% confidence interval How is it demonstrated? Patient serum MUST be: Positive with 3 cells with the antigen Negative with 3 cells without the antigen

38. Our previous example fulfills the “rule of three”

39. What if the “rule of three” is not fulfilled? If there are not enough cells in the panel to fulfill the rule, then additional cells from another panel could be used Most labs carry different lot numbers of panel cells (or screening cells)

40. Phenotyping In addition to the rule of three, antigen typing the patient red cells can also confirm an antibody How is this done? Only perform this if the patient has NOT been recently transfused (donor cells could react) If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?

41. Remember Landsteiner’s Rule?

42. MULTIPLE ANTIBODIES Identifying multiple antibodies may be more of a challenge than a single antibody Why? Reaction strengths can vary Matching the pattern is difficult

43. So what is a tech to do? Several procedures can be performed to identify multiple antibodies Selected Cells Neutralization Adsorption Chemical treatments

44. Selected Cells Selected cells are chosen from other panel or screening cells to confirm or eliminate the antibody The cells are “selected” from other panels because of their characteristics The number of selected cells needed depends on how may antibodies are identified

45. Selected Cells Every cell should be positive for each of the antibodies and negative for the remaining antibodies For example: Let’s say you ran a panel and identified 3 different antibodies: anti-S, anti-Jka, and anti-P1 Selected cells could help…

46. Selected Cells These are panel cells that are being used, I just didn’t include the rest of the antigram because it is not necessary.These are panel cells that are being used, I just didn’t include the rest of the antigram because it is not necessary.

47. Neutralization Some antibodies may be neutralized to separate antibodies or confirm the presence of an antibody Commercial “substances” bind to the antibodies in the patient serum, causing them to show no reaction when tested with the corresponding antigen (in panel)

48. Neutralization Manufacturer’s directions should be followed and a dilutional control should always be used The control contains saline and serum (no substance) and should remain positive (nothing is being neutralized) A control shows that a loss of reactivity is due to the neutralization and not to the dilution of the antibody strength when the substance is added

49. Neutralization Common substances P1 substance (sometimes derived from hydatid cyst fluid) Lea and Leb substance (soluble antigen found in plasma and saliva) I substance can be found in breast milk Sda substance derived from human or guinea pig urine **you should be aware that many of these substances neutralize COLD antibodies; Cold antibodies can sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization techniques

50. Adsorption Adsorption procedures can be used to investigate underlying alloantibodies especially when the patient has an autoantibody to common antigens (e.g., I, H) ZZAP or chloroquine diphosphate can be used to dissociate IgG antibodies from the RBC (may take several repeats) After RBCs are incubated with patient serum (to adsorb the autoantibody), the residual serum is tested with panel cells to ID the alloantibody (if present)

51. Adsorption Two types: Autoadsorption No recent transfusion Autoantibodies are removed using patient RBCs, so alloantibodies in serum can be identified using a panel Allogenic (Differential) adsorption If recently transfused (past 3 months) Uses other cells with the patients serum

53. Enzymes (proteolytic) Can be used to enhance or destroy certain blood group antigens Several enzymes exist: Ficin (figs) Bromelin (pineapple) Papain (papaya) In addition, enzyme procedures may be One-step Two-step

54. Enzymes Enzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other antigens to be “enhanced” Antigens destroyed: M, N, S, s, Duffy Antigens enhanced: Rh, Kidd, Lewis, I, and P

55. Enzyme techniques One-stage Enzyme is added directly to the serum/cell mixture Two-stage Panel cells are pre-treated with enzyme, incubated and washed Patient serum is added to panel cells and tested

56. Enzyme techniques If initial agglutination disappears after treatment, then it is assumed the enzymes destroyed the antigen Duffy, M, N, S, s If agglutination is stronger, the enzymes enhanced the antigen Rh, Kidd, Lewis, P Some antigens are not affected Kell, U, Lutheran

58. Other chemicals: Sulfhydryl Reagents Cleave the disulfide bonds of IgM molecules and help differentiate between IgM and IgG antibodies Good to use when you have an IgM and need to an ID underlying IgG, usually after enzyme treatment Both reagents denature K antigens Jsa and Jsb are particularly vulnerable Dithiothreitol (DTT) — a thiol 2-mercaptoethanol (2-ME)

59. Effect of enzymes and DTT

60. Other Chemicals:AET 2-aminoethylisothiouronium bromide (2-AET) Removes activity of Kell system antigens from RBCs (except Kx)

61. Other Chemicals:ZZAP A combination of proteolytic enzymes (papain) and DTT Destroys Kell, M, N, S, Duffy and other less frequent blood group antigens Does not denature Kx antigen Dissociates IgG bound to RBCs Good for adsorption techniques Used more when warm autoantibodies are present

62. Other Chemicals:CDP and Glycine acid EDTA Both of these chemicals dissociate IgG from RBCs They render DAT+ red cells DAT- Keeps the RBC intact for phenotyping (using indirect AHG phase)

63. Other Chemicals: Chloroquine diphosphate: Dissociates IgG from RBC May weaken Rh antigens Denatures HLA antigens (Bg) on RBCs RBCs should be treated for about an hour (no more than 2) Glycine Acid EDTA: Dissociates IgG from RBC Takes only a few minutes compared to an hour to treat RBCs Destroys Kell, Cartwright (Cw), Er, Bg Useful in denaturing antigens for select cell panels

64. Special Considerations in Antibody ID

65. Getting a positive DAT We have focused a lot on the IAT used in antibody screening and ID, but what about the DAT? The direct antiglobulin test (DAT) tests for the in vivo coating of RBCs with antibody or complement (in the body) AHG is added to washed patient red cells to determine if IgG or complement are coating the RBCs

66. What can the DAT tell us? Although not always performed in routine pretransfusion testing, a positive DAT can offer valuable information If the patient has been recently transfused, the patient may have an alloantibody coating the transfused cells If the patient has NOT been transfused, the patient may have an autoantibody coating their own cells A positive DAT may also occur when a red top is used (in vitro bound complement)

67. Autoantibodies Autoantibodies can be cold or warm reacting A positive autocontrol or DAT may indicate that an auto-antibody is present Sometimes the autocontrol may be positive, but the antibody screen may be negative, meaning something is coating the RBC

68. Identifying autoantibodies Auto-antibodies can sometimes “mask” clinically significant allo-antibodies, so it’s important to differentiate between auto- and allo-antibodies

69. Cold autoantibodies Many adults have low titers of autoanti-I, -H, and –IH Since they are IgM, they are not significant Cold autoantibodies should be suspected when all of the screening cells, panel cells and AC are positive at IS and get weaker with incubation; some can fix complement and will agglutinate with poly AHG Could be triggered by Mycoplasma pneumoniae or infectious mono

70. Example

71. Resolution: Cold autoantibodies Cold autoantibodies can be a nuisance at times. Here are a few ways to avoid a reaction: Prewarmed techniques Red cells, serum, and saline are incubated at 37° before being combined Autoadsorption is another technique in which the autoantibody is removed from the patients serum using their own red cells The serum can be used to identify any underlying alloantibodies

72. Resolution: Cold autoantibodies Sulfhydryl compounds DTT and 2-ME break the disulfide bonds in IgM The patient’s serum is treated with the compound before antibody screening or ID

73. Cold autoantibodies Cold antibody screen can be used to help identify cold autoantibodies Since anti-I is a common autoantibody, cord blood cells (no I antigen) are usually included

74. Warm autoantibodies More common that cold autoantibodies Positive DAT due to IgG antibodies coating the red cell Again, the majority of panel or screening cells will be positive It is not necessary to ID the autoantibody, although many are Rh antibodies (especially anti-e)

75. Warm autoantibodies Cause warm autoimmune hemolytic anemia (WAIHA)…?H&H How do you get a warm autoantibody? Sometimes idiopathic 2° to CLL, lymphoma, SLE, autoimmune… Medications Several techniques are used when warm autoantibodies are suspected, but the main goal is to determine any underlying alloantibodies

76. Example

77. Resolution: Warm Autoantibodies Autoadsorption is the method of choice (see previous slide) unless patient has recently been transfused Partial elution to remove Ab (ZZAP) Cells are then used to adsorb Ab from serum and serum is used for alloantibody testing If no clinically significant Ab are detected, and IS crossmatch is all that is needed

78. Antibodies to High Frequency Antigens High frequency antigens are present in most individials (>98%) Suspect when most or all screen and panel cells are positive with negative autocontrol Examples: U and HTLA (Ch, Rg) Autologous donations encouraged due to difficulty finding compatible blood

79. Antibodies to Low Frequency Antigens Low frequency antigens occur in <10% of the population Antibodies are uncommon, but should be suspected when the crossmatch is incompatible and no problems occur with ABO or DAT The antibody panel may only have 1 cell react Resolution: test with additional antigen positive cells to confirm

80. Elution Elutions are performed whenever a DAT is positive Most often performed when transfusion reactions are suspected or if HDN is suspected

81. Elution (whenever DAT is positive) Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified

82. Elution The eluate is a term used for the removed antibodies Testing the eluate is useful in investigations of positive DATs HDN Transfusion reactions Autoimmune disease The red cells can also be used after elution for RBC phenotyping if needed When tested with panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present

83. Elution Acid elutions (glycine acid) Most common Lowers pH, causing antibody to dissociate Organic solvents (ether, chloroform) Dissolve bilipid layer of RBC Heat (conformational change) Freeze-Thaw (lyses cells)

84. Elution Many of elution tests can damage the antigens on the RBC Choroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate IgG from the RBC without damaging the antigens Very useful if the RBC needs to be antigen typed

85. THE END